A salicyl fumarate derivative and its application in the treatment of parkinson&#39;s disease and other neurodegenerative diseases

ABSTRACT

A salicyl fumarate derivative, and its general structural formula (A) is: 
     
       
         
         
             
             
         
       
     
     Wherein the structural formula (A), R 1  is one of H + , Na + , K +  or NH4 + . R 2  is one of fumaric acid ester products. It has the general structural formula of the combination of salicylic acid and fumaric acid through esterification reaction. This category of compounds possesses good effects in treatment of neurodegenerative diseases.

TECHNOLOGY AREA

This invention provides a new class of compounds, more specificallyrelates to a salicyl fumarate derivative, and its application in thetreatment of Parkinson's Disease (referred to as PD hereafter).

TECHNOLOGY BACKGROUND

Neurodegenerative disease refers to a category of diseases in whichneurons in the brain and the spinal cord are progressively lost. Thebrain and the spinal cord are made up of neurons, which have differentfunctions, such as controlling movement, handling sensory information,and making decisions. Cells in the brain and spinal cord are generallynot regenerated, so excessive damage on the neurons can be devastatingand irreversible. Neurodegenerative diseases are caused by the loss ofneurons or their myelin sheath, which worsens over time to lead todysfunction. Neurodegenerative diseases mainly manifest into twoaspects: one is movement disorder, such as cerebellar ataxia, the otheris memory dysfunction and its related dementia.

As aging in population intensifies, the rate of occurrence ofneurodegenerative diseases is also climbing. Neurodegenerative diseaseis a diseased state in which neurons in the brain and spinal cord arelost, where neuronal cells generally do not regenerate. Excessive damagecan be devastating and irreversible. Neurodegenerative diseases worsenover time, eventually leading to dysfunction. Alzheimer's Disease andParkinson's Disease are the two diseases with the highest prevalence,causing great pain to patients as well as their families.

Previous studies have shown that neurons can absorb injected α-synucleinproteins in both in vitro and in vivo models to form Lewy body proteinswhich are PD's characteristics. In 2014 Recasens et al. published thatwhen Lewy bodies extracted from the substantia nigra of PD patients wereinjected into the substantia nigra or striatum of mice and rhesusmonkeys, α-synuclein protein accumulated in host neurons. After 4-17months, the dopaminergic neuron terminals appeared neurodegenerative,similar to PD pathology. They published the findings in Annals ofNeurology (2014) (Publication information: Recasens A, Dehay B, Bove J,et al. Lewy body extracts from Parkinson disease brainstrigger-synuclein pathology and neurodegeneration in mice and monkeys.Annals of Neurology. 2014, 75:351-362.)

In the same year, Unterberger et al. proposed a new mechanism, which ishow α-synuclein spreads amongst neurons in PD. This team used anantibody that can distinguish between normal and abnormal α-synuclein inorder to confirm how the pathological protein migrate to neighboringneurons. The same team also used antibodies to detect abnormalα-synuclein proteins in cerebrospinal fluid. This method will help earlyPD diagnosis and diagnoses of other α-synuclein diseases. It waspublished in Neurobiology of Disease (2014) (Publication information:Unterberger U, Lachmann I, Voitlander T. et al. Detection ofdisease-associated alpha-synuclein in the cerebrospinal fluid: afeasibility study. Neurobiology of Disease. 2014, 33:329-334.)

Chao J provided a technical scheme concerning a type of fumaric acid andits derivative, and acquired American patent authorization (BulletinNo.: US20140194427A1). In the technical scheme, the fumaric acidderivative provided by Chao J had a specific structural formula as shownin Figure (I). Chao J claimed the demonstrated fumaric acid derivativehas treatment properties against neurodegenerative diseases, such asmultiple sclerosis (MS), amyotrophic lateral sclerosis, PD, Huntington'sDisease, or Alzheimer's Disease.

The USA FDA has approved a new twice-daily orally administered drugdimethyl fumarate (DMF, commercially known as Tecfidera) to be used totreat relapsing-remitting multiple sclerosis (RRMS). DMF has become oneof the many approved treatments for MS. According to the NationalMultiple Sclerosis Society archives, two dosage forms of Interferon β-1a(Avonex and Rebif) as well as two dosage forms of β-1b (Betaseron andExtavia) have already been approved for RRMS therapy. Other drugsapproved include Glatiramer acetate (Copaxone), Fingolimod (Gilenya) andTeriflunomide (Aubagio). Natalizumab (Tysabri) has received approval aswell, but with conditional restrictions. Mitoxantrone (Novantrone) isapproved to treat secondary progressive, progressive recurrent, andrecurrent remission types of MS. Dalfampridine (Ampyra) is approved forimproving the movement of MS patients.

Biogen Idec claims that, in the study called DEFINE, DMF users in MSpatients have shown a 49% and 38% reduction in occurrences of recurrenceand disability, while in the study called CONFIRM, DMF users have showna 34% reduction in recurrence. Both studies illustrate that, compared toplacebo, DMF can significantly reduce brain injury. Dr. Robert Fox fromCleveland Clinic Multiple Sclerosis Mellen Center claimed in a BiogenIdec report: “In clinical trials, compared to the placebo group, diseaseactivity in DMF patients decreases, whether they are early-stage MSpatients or more progressed patients. This drug provides doctors withanother important treatment option to target MS patients of differentstages.”

However, FDA notes that DMF can lower the lymphocyte counts, thoughthere is no evidence to suggest an increase in infections in DMF users.They suggest monitoring patient lymphocyte counts before and aftertreatment. Reddened face and gastrointestinal distress are the mostcommon side effects.

Ahuja M et al. demonstrated the effect of DMF and its active metabolitemonomethyl fumarate (MMF) on the Nrf2 signaling pathway as well as on1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse PDmodels. Their study confirmed that DMF and MMF exhibit protectiveeffects against the neural toxicity of MPTP in mouse PD models. This isdue to their unique Nrf2-mediated anti-oxidant, anti-inflammatory, andenhancement of mitochondrial functions. Unlike DMF, MMF does not consumeglutathione and inhibit mitochondrial and glycolysis functions. Thepathogenesis of PD includes oxidative damage, inflammation, andmitochondrial dysfunction. Ahuja M et al. claimed that MMF is the trueactive ingredient for treating PD. Their findings were published in TheJournal of Neuroscience in 2016 (Publication information: Manuj Ahuja,Navneet Ammal Kaidery, Lichuan Yang, Noel Calingasan, Natalya Smirnova,Arsen Gaisin, Irina N. Gaisina, Irina Gazaryan, Dmitry M. Hushpulian,Ismail Kaddour-Djebbar, Wendy B. Bollag, John C. Morgan, Rajiv R. Ratan,Anatoly A. Starkov, M. Flint Beal, and Bobby Thomas. Distinct Nrf2Signaling Mechanisms of Fumaric Acid Esters and Their Role inNeuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-TetrahydropyridineInduced Experimental Parkinson's-Like Disease. The Journal ofNeuroscience. 2016, 36(23): 6332-6351.)

Content of Invention

Based on early research discoveries and considering the side effects ofDMF, the applicants invented a brand-new class of compounds and verifiedgood activity the compounds have on Parkinson's disease (PD) models,which may also apply to other neurodegenerative diseases such asAlzheimer's disease (AD), and Huntington's disease (HD).

The new compounds provided by the applicants are salicyl fumaratederivatives. Its general structural formula (A) is:

The R₁ in the structural formula (A) is one of H⁺, Na⁺, K⁺ or NH4, R₂ isa fumaric acid ester.

The applicants further confirmed in their study that the above-mentionedstructural formula, the fumaric acid ester of the R₂ group is one ofmethyl fumarate, mono-ethyl fumarate, mono-propyl fumarate,mono-isopropyl fumarate, mono-butyl fumarate, or mono-tertbutylfumarate.

On the premise to determine the R₂ group, the applicants furtherconfirmed that the salicyl fumarate derivative is methyl salicylfumarate, the structure of which is:

On the premise to determine the R₂ group, the applicants furtherconfirmed that the salicyl fumarate derivative is ethyl salicylfumarate, the structure of which is:

For all of the above salicyl fumarate derivatives, the R₁ group may beone of H⁺, Na⁺, K⁺ or NH4⁺. This depends on whether the applicants inaim of specific use of salicyl fumarate derivatives need to use thereaction between alkaline compounds and salicyl fumarate derivatives togenerate salt forms. If the applicants do not use alkaline compounds toreact with the salicyl fumarate derivative, then the R₁ will be a H⁺. Ifthe reaction is with NaOH solution, then R₁ group will be a Na⁺. Theother changes to the R₁ group are similar situations. Current researchfrom the applicants show no direct effect from substituting R₁ group onthe activity of salicyl fumarate derivative. Nonetheless, substitutingR₁ changes the solubility of the salicyl fumarate derivative, thereforemay affect the absorbability of salicyl fumarate derivative by thegastrointestinal tract.

As for the methodology for production of salicyl fumarate derivative,this method is carried out by esterification reaction. But becausesalicylic acid has two groups, hydroxyl and carboxyl groups, and undernormal conditions, the stability of the carboxyl group is poorer; inorder to achieve chemical directional reaction, the applicants designedthe experiment to first use the esterification reaction of tert-butanolwith salicylic acid to protect the carboxyl group. Then the productreacts with the mono-esterified fumarate. Finally, the target product isobtained by removing tert-butanol. The applicants provide methodologythat includes the following steps:

1) The esterification reaction of tert-butanol and salicylic acid iscarried out to protect the carboxyl group of salicylic acid to obtainintermediate product I;

2) The esterification reaction of intermediate product I and the fumaricacid derivative is carried out to obtain intermediate product II;

3) Remove tert-butanol from intermediate product II under acidiccondition to obtain the target product III.

The chemical reaction formulae are as follows:

Through pharmacological experiments, the applicants demonstrated theapplication of salicyl fumarate derivatives as new therapeutic compoundsin PD treatment.

The technology beneficial effect of the invention is: The presentinvention produces a salicyl fumarate derivative, which contains thegeneral structural formula combining salicylic acid and fumaratederivative through esterification. The applicants throughpharmacological experiments have verified that this kind of compoundshas protective activity in the treatment of neurodegenerative diseases.

DESCRIPTION OF ATTACHED FIGURES

FIG. 1 MR spectrogram of Compound A (methyl salicyl fumarate);

FIG. 2 MR spectrogram of Compound B (ethyl salicyl fumarate);

FIG. 3 Dose response protection and safety study of MSF and ESF onMPTP-induced model of dopaminergic neuronal damage in mice;

FIG. 4 Protective effects of MSF and ESF on dopaminergic neurons inMPTP-induced model of chronic dopaminergic neuronal damage in mice;

FIG. 5 Effects of MSF and ESF on the concentrations of MPTP metabolitesMPP+;

FIG. 6 HPLC Chromatogram of MSF in mouse blood sample;

FIG. 7 HPLC Chromatogram of ESF in mouse blood sample;

FIG. 8 Mouse blood drug concentrations with MSF and ESF dosage of 100mg/kg;

FIG. 9 Mouse liver drug concentrations with MSF and ESF dosage of 100mg/kg;

FIG. 10 Mouse brain drug concentrations with MSF and ESF dosage of 100mg/kg;

FIG. 11 Under different MSF and ESF dosages, the drug concentrations inmouse striatum;

FIG. 12 The protective effect on dopaminergic neurons in Nrf2 knockoutmice with MPTP-induced dopaminergic neuronal damage;

FIG. 13 Effects of MSF and ESF on intracellular glutathioneconcentrations in in vitro experiments;

In above figures, FIGS. 1 and 2 are NMR spectrograms generated byinstrument work station; they were originally in color. After they wereadjusted to black and white there were some text-and-image overlaps.This specific information has been documented in the ImplementationExample 1 in the next section;

Chinese to English translations for FIGS. 3, 4, 5, 12, and 13;

Chinese to English translations for FIGS. 6 and 7;

Chinese to English translations for FIGS. 8, 9, and 10.

Specific Implementation Patterns

Implementation Example 1 Synthesis of Salicyl Fumarate Derivatives

1. Protecting the Carboxyl Group on the Salicylic Acid

Dissolve salicylic acid (45 g, 795.9 nmol, 1 equivalent weight) indimethylformamide (DMF) (450 ml), under 0° C. (ice water bath)condition, add N′N-carbonyldiimidazole (63.5 g). At room temperature mixfor 1 h, and at the same time slowly drip DBU (58.5 ml) and tert-butanol(63 ml). Then at room temperature mix for 2 h. After the reaction isdone use LC-MS to detect (product spectrogram is illustrated in attachedFIG. 1). Put reactants into water (500 ml), and use ethyl acetate toextract 3 times (3×800 ml). Combine with organic layers, then rinse 3times (3×800 ml) with water; then rinse 1 time with saline (1000 ml);then dry with anhydrous Na₂SO₄; then filter; then concentrate thefiltrate until no organic solvent residue is left. Purify the producedconcentrate by silica gel column chromatography (petroleum ether:ethylacetate=100:1 as mobile phase), obtaining the product (product 52 g,yield 82.5%). Product is light-yellow and oil-like (named: chemicalcompound A3). The chemical reaction formula is as follows:

2. Esterification of Monomethyl Fumarate with Compound A3 (Compound A4)

Add DCM (100 ml) to monomethyl fumarate (10.4 g), then add the mixedsolution of HATU (45.6 g) and DIEA (31 g). The reaction mixture isstirred for 30 minutes, then compound A3 (10 g) is added to the reactionmixture at room temperature and stirred for 16 hours at roomtemperature, LC-MS shows the reaction is complete. The reaction mixtureis quenched with water (200 mL) and extracted with DCM (2×200 mL). Washthe combined organic layer with salt water (300 mL), then dry withanhydrous Na₂SO₄; then filter; then concentrate the filtrate until noorganic solvent residue is left. Purify the produced concentrate bysilica gel column chromatography (petroleum ether:ethyl acetate=30:1 asmobile phase), obtaining the product (product 12.6 g, yield 79.9 g).Product is light-yellow and oil-like (named: chemical compound A4). Thechemical reaction formula is as follows:

3. Preparation of Methyl Salicyl Fumarate (Compound A)

Under 0° C. (ice water bath) conditions, add compound A4 (5.6 g) tomixed solution of DCM (100 ml), 2N HCl and Et₂O (25 ml). The mixturereacts by stirring at room temperature overnight, TCL shows the reactionis complete. Remove the reaction solvent, add water (100 ml), thenextract 3 times with DCM (3×150 ml). Combine organic layers, then rinsewith salt water; dry with anhydrous Na₂SO₄. The product is purified bysilica gel column chromatography (petroleum ether:ethyl acetate=2:1),and crude product is obtained. Mix the crude product with n-hexane(crystallization) overnight, filter to obtain pure product A (product4.5 g, yield 98.4%); analyze with MR and MS assays, pure product A isidentified as methyl salicyl fumarate. The reaction formula is asfollows:

4. Esterification of Mono-Ethyl Fumarate with Compound A3 (Compound B2)

Add mono-ethyl fumarate (10.4 g) to DCM (100 ml), then add HATU (45.6 g)and DIEA (31 g) to mixed solution. The reaction mixture is stirred for30 minutes, then compound A3 (10 g) is added to the reaction mixture atroom temperature and stirred for 16 hours at room temperature, LC-MSshows the reaction is complete. The reaction mixture is quenched withwater (200 mL) and extracted with DCM (2×200 mL). Wash the combinedorganic layer with salt water (300 mL), then dry with anhydrous Na₂SO₄;then filter; then concentrate the filtrate until no organic solventresidue is left. Purify the produced concentrate by silica gel columnchromatography (petroleum ether:ethyl acetate=30:1 as mobile phase),obtaining the product (product 12.8 g, yield 79.9%). Product islight-yellow and oil-like (named: chemical compound B2). The chemicalreaction formula is as follows:

5. Preparation of Ethyl Salicyl Fumarate (Compound B)

Under 0° C. (ice water bath) conditions, add compound B2 (5.6 g) tomixed solution of DCM (100 ml), 2N HCl and Et₂O (25 ml). The mixturereacts by stirring at room temperature overnight. TCL shows the reactionis complete. Remove the reaction solvent, add water (100 ml), thenextract 3 times with DCM (3×150 ml). Combine organic layers, then rinsewith salt water; dry with anhydrous Na₂SO₄. The product is purified bysilica gel column chromatography (petroleum ether:ethyl acetate=2:1),and crude product is obtained. Mix the crude product with n-hexane(crystallization) overnight, filter to obtain pure product A (product4.0 g, yield 86.5%); analyze with MR and MS assays, pure product B isidentified as ethyl salicyl fumarate. The reaction formula is asfollows:

6. Analyzing compound A and compound B, the specific data are below:

MR Spectrometry Assay

Compound A: ¹H NMR (400 MHz, DMSO): δ13.18 (s, 1H), 7.98 7.96 (m, 1H),7.71˜7.67 (m, 1H), 7.46˜7.42 (m, 1H), 7.32˜7.30 (m, 1H), 6.99˜6.98 (m,2H), 3.80 (s, 3H); (NMR chromatogram shown in FIG. 1). MR spectrometryverifies compound A as methyl salicyl fumarate.

Compound B: ¹H NMR (400 MHz, DMSO): δ13.16 (s, 1H), 7.98 7.96 (m, 1H),7.70 7.67 (m, 1H), 7.45˜7.41 (m, 1H), 7.31˜7.29 (m, 1H), 6.97˜6.96 (m,2H), 4.27˜4.22 (m, 2H), 1.29˜1.26 (m, 3H); (NMR chromatogram shown inFIG. 2). MR spectrometry verifies compound B as ethyl salicyl fumarate.

7. The applicants used the same fore-mentioned synthesis routes toperform esterification of salicylic acid and fumarate esters. Theyobtained propyl salicyl fumarate (compound structural formula shown asC), isopropyl salicyl fumarate (compound structural formula shown as D),and butyl salicyl fumarate (compound structural formula shown as E)respectively. Considering the pharmacological activities of propylsalicyl fumarate, isopropyl salicyl fumarate, and butyl salicyl fumaratewere lower than those of methyl salicyl fumarate and ethyl salicylfumarate through preliminary experiments, the pharmacological studies ofthese three compounds will not be mentioned in this application.

However, from the synthesis routes of the 5 compounds, methyl salicylfumarate, ethyl salicyl fumarate, propyl salicyl fumarate, isopropylsalicyl fumarate, and butyl salicyl fumarate, it can be known that ifthe carboxyl group on the salicylic acid can be effectively protected,salicyl fumarate derivatives can be synthesized. Therefore it isverified that the synthesis routes provided by the applicants aregenerally applicable for the effective synthesis of salicyl fumaratederivatives.

Implementation Example 2 the Protective Role of MSF and ESF onDopaminergic Neurons

Using MSF and ESF to conduct therapeutic studies with widely acceptedMPTP-induced dopaminergic neuron-damaged PD mouse models. Dimethylfumarate (DMF) was used as positive control drug, 6 dose groups wereused for DMF. For the experimental groups of MSF and ESF, 10 dose groupswere used for each drug. 10 mice in each group. Both the experimentalgroup and the control group used intraperitoneal administration. Thecontrol group was treated with DMF. The experimental groups were treatedwith different doses of ESF and MSF (as shown in FIG. 3). Statisticallysignificant protective effects of dopamine neurons were found, and thedose-effect relationship was observed. The lowest dosage for protectiveeffect (10 mg/kg/d), the optimal dosage (100-200 mg/kg/d), and thedosage where toxic effects began to appear (>500 mg/kg/d) weredemonstrated. Compared to the results of DMF, MSF and ESF had moresignificant protective effects. Furthermore, the dosage where DMF showedprotective effect (50 mg/kg/d) was higher than MSF and ESF, and thedosage where DMF showed toxic effect (>200 mg) was lower than MSF andESF. Therefore, MSF and ESF have more effective and safer therapeuticdose windows (as shown in FIG. 3).

MSF and ESF (200 mg/kg/d) also showed significant dopamine neuronprotection in models of chronic MPTP-induced dopamine neuron damage inmice, and the protection of dopamine neurons was close to a blankcontrol level (as shown in FIG. 4).

The concentration of MPTP metabolite MPP+ in brain tissue was determinedby HPLC. For the MPTP injected mouse model, the ESF (500 mg/kg)administered group and the blank group had the same MPP+ content in thestriatum, excluding the possibility that MSF and ESF had an effect onthe uptake and metabolism of MPTP in vivo and in the brain, confirmingtheir neuroprotective effects (as shown in FIG. 5).

A reliable, high-performance liquid chromatography MSF and ESF analysismethod (as shown in FIGS. 6 and 7) was established to determine thelevel of MSF and ESF in peripheral blood, liver, and brain tissue,demonstrating that MSF and ESF can enter the brain through theblood-brain barrier after intraperitoneal injection in mice.Furthermore, the molecular structure of MSF and ESF remains unchanged inthe brain (as shown in FIG. 8, FIG. 9, FIG. 10, and FIG. 11), and it isinferred that MSF and ESF molecules produce neuroprotectivepharmacological activities directly through their original moleculestructures. Unlike the positive control drug DMF which workeddifferently. DMF needs to entering the body and quickly metabolizinginto a form of single methyl metabolite monomethyl fumarate (MMF) whichis the active form to produce pharmacological activity.

MSF and ESF have conventional fumaric acid (fumarate) activation targetNrf2 similar to the positive control drug DMF, which results inactivating the mechanism of intracellular antioxidative stress. Byperforming MPTP experiments on transgenic mice with the Nrf2 geneknocked out, DMF lost the dopaminergic neuron protective effect due tothe loss of its effective target, while MSF and ESF still showsignificant protective effects in this model (as shown in FIG. 12); thusconfirming that MSF and ESF, in addition to acting on Nrf2 target, alsoact on another neuroprotective target. Inferring from the inclusion ofsalicyl in their molecule structure, this new target may be associatedwith anti-neuroinflammation.

The results of in vitro cell experiments showed that positive controldrug DMF had the effect of depleting glutathione (GSH, endogenousantioxidant polypeptide), which may be the cause of its obvious toxicityand side effects. MSF and ESF have no effect on the concentration ofglutathione in the cell (as shown in FIG. 13).

1. A salicyl fumarate derivative wherein: its general structural formula(A) is:

Within the structural formula (A), R₁ is one of H⁺, Na⁺, K⁺ or NH4⁺, R₂is one of fumaric acid ester products.
 2. The salicyl fumaratederivative according to claim 1, wherein: the esterification of thefumaric acid is one of: mono-methyl fumarate, mono-ethyl fumarate,mono-propyl fumarate, mono-isopropyl fumarate, mono-butyl fumarate,mono-tertbutyl fumarate as described.
 3. The salicyl fumarate derivativeaccording to claim 2, wherein: the stated salicyl fumarate derivative ismethyl salicyl fumarate, its structural formula is:


4. The salicyl fumarate derivative according to claim 2, wherein: thestated salicyl fumarate derivative is ethyl salicyl fumarate, itsstructural formula is:


5. The production method for the described salicyl fumarate derivativeaccording to claim 1, wherein: 1) The esterification reaction oftert-butyl alcohol and salicylic acid is carried out, protecting thecarboxyl group of salicylic acid, obtaining intermediate product I; 2)The esterification of intermediate product I and the fumaric acidderivative is carried out, obtaining intermediate product II; 3) Thetert-butanol in intermediate product II is removed under acidiccondition, obtaining target product III. The chemical reaction formulais as follows:


6. A method comprising applying the salicyl fumarate derivativeaccording to claim 1 to drug production for the treatment of Parkinson'sDisease.
 7. A method comprising applying the salicyl fumarate derivativeaccording to claim 2 to drug production for the treatment of Parkinson'sDisease.
 8. A method comprising applying the salicyl fumarate derivativeaccording to claim 3 to drug production for the treatment of Parkinson'sDisease.
 9. A method comprising applying the salicyl fumarate derivativeaccording to claim 4 to drug production for the treatment of Parkinson'sDisease.